: Checkpoints assure that progression from one cell cycle stage to the next proceeds only after previous events have been completed. The long-term goal is to elucidate the molecular mechanism of checkpoints activated by DNA damage in S phase, which results in down-regulation of DNA synthesis. The hypothesis to be addressed by this proposal is that radiation induces a signal transduction pathway that stalls DNA replication by inactivating factors involved in replication initiation. The goal is to identify the replication machinery targets and use the information gained to define the signal transduction pathway. An SV40 in vitro replication system will be used. All components needed have been purified and the SV40 large T antigen is the only non-cellular component. The specific aims include: 1) to study the post-translational modification and activity of RP-A; 2) to study the post-translational modification and activity of SV40 T antigen; 3) to study the contribution of ATM, p53 and p21 to signalling that can lead to modifications of RP-A and SV40 T antigen; and 4) to begin to purify novel proteins involved in signalling.